1. Field of the Invention
The present invention relates to an amidite for synthesizing modified nucleic acid which is preferably used for producing a hydroxyl group-containing modified nucleic acid, and a method for synthesizing modified nucleic acid using the amidite.
2. Description of the Related Art
Unraveling of the whole human genome has shifted the focus of interest of scientists and researchers on the analysis of proteins, which are gene products. It may not be too much to say that substantial protein analysis can be made possible only when a molecule that shows affinity for a protein of interest has been successfully obtained. A cell, however, contains many different types of proteins, and the amino acid sequence and structure of many of which are still unknown.
The most common technique for obtaining a molecule that shows affinity for a specific protein is to prepare an affinity antibody by utilizing the immune system of animal. However, this technique uses animals and requires a large quantity of proteins, a large number of processes and large costs. Additionally, no affinity antibody may be obtained for specific substances with this technique.
A technique called aptamer method (also referred to as SELEX) that does not rely on any living organism has been proposed to avoid this problem. However, while a molecule obtained by this technique strongly interacts with a specific protein, this technique is not applicable to all proteins. In view of the above-identified circumstances, the inventors proposed a modified aptamer method that is established by improving the aptamer method so as to use modified nucleic acid (see International Publication No. WO2003/078623).
Solid-phase synthesis of nucleic acids was initiated as long ago as more than 20 years, and automatic synthesizers were already sold then. Solid-phase synthesis of nucleic acid is carried out, for example by making a nucleic acid material (amidite) combine in a condensation reaction with a solid carrier (e.g. CPG) in which a nucleoside is immobilized; this condensation reaction needs to take place, with only a phosphoric acid portion in the amidite and a hydroxyl group in another amidite being involved in the condensation reaction, and other reactive groups not being involved in the condensation reaction. Therefore, it is necessary to prevent an exocyclic amino group, etc. contained in a base of an amidite used from being involved in the condensation reaction by introducing a protective group, and to eliminate (remove) the protective group after the condensation reaction has finished completely. Conventionally, benzoyl group, isobutyryl group and the like have been used for protective groups introduced into exocyclic amino groups in bases, and a method of allowing concentrated ammonia water to act at 55° C. for 8 hours to 15 hours so as to remove these protective groups has been common.
However, when a modified nucleic acid that shows affinity for a protein as described above is produced under the above-described conventional deprotection conditions, a modified moiety in the modified nucleic acid (a substituent showing affinity for a protein) is removed together with a protective group. Thus, modified nucleic acids can not be stably produced. Therefore, when the modified nucleic acids are to be produced, amidites which enable protective groups therein to be removed under more moderate conditions are desired to avoid removal of substituents which show affinity for a protein together with protective groups.
For example, in the related art, nucleic acid amidites which enable protective groups therein to be removed by diazabicycloundecene (DBU) that is a bulky base (refer to Acta Chem, Scand., B37, 263 (1983) and J. Org. Chem., 54, 1657 (1989)) have been reported. However, they are not suitable for practical use, since the amidites for synthesizing nucleic acid represented are unstable in acetonitrile that is an aprotic solvent (refer to Tetrahedron Letters 46, 6729 (1990)). Additionally, although it has also been reported that amidites for synthesizing nucleic acid enable protective groups therein to be removed in pyridine under a condition of 0.5M DBU for 16 hours (refer to Tetrahedron 40, 4171 (1992) and Nucleodied & Nuclrotides 13, 2059 (1994)), they are problematic in that nucleic acid bases are alkylated owing to highly-concentrated DBU and deprotection for a long period of time. In addition, although it has also been reported that amidites for synthesizing nucleic acid represented enable protective groups therein to be removed using K2CO3 in methanol (refer to Tetrahedron Letters 46, 6729 (1990) and Nucleic Acids Research 21, 3493 (1993)), they are problematic in that esters, etc. decompose because K2CO3 that is a base is used in methanol that is a protic solvent.
Thus, as things stand at present, development of an excellent amidite for synthesizing modified nucleic acid which enables a protective group therein to be removed under a moderate condition, thereby stably producing modified nucleic acid that can be suitably used for analysis of a target substance such as a protein, and of a method for synthesizing modified nucleic acid using the amidite for synthesizing modified nucleic acid is still hoped for.